Several histochemical staining protocols, including Hematoxylin and Eosin (H&E) staining and Papanicolaou (PAP) staining, rely on the dye hematoxylin to stain cytological and tissue samples. In particular, hematoxylin staining of cell nuclei is used by pathologists to detect the presence of malignant and/or metastatic cells in a tumor biopsy sample.
Hematoxylin is a naturally-occurring compound found in the red heartwood of trees of the genus Hematoxylon. Hematoxylin itself is colorless in aqueous solution and is not the active ingredient that stains tissue components. Rather, an oxidation product of hematoxylin, hematein, becomes the active staining component of a hematoxylin dye solution, particularly upon forming a complex with a mordant. Hematein is produced naturally through exposure to air and sunlight. The natural process is termed “ripening”, and can take 3 or more months to provide a solution suitable for staining cells.
Automated staining procedures and systems use mechanical systems to deliver staining solutions to a biological sample. Standard hematein staining procedures utilized a premixed stock containing both the hematoxylin-hematein and a mordant. Precipitates form in these premixed stocks. This is not generally a problem for manual staining procedures, where slides are treated with the hematoxylin staining solution in a container, such as a glass container. However, precipitates are a problem for automated staining systems where the precipitate can foul or clog delivery lines and make cleaning or purging of the delivery lines difficult. These changes to hematoxylin and the precipitates in staining solutions can result in staining inconsistencies. For example, hematoxylin stain stocks containing mordant are often allowed to ripen for an extended period of time, allowing developing of hematein-mordant complexes. While this process may allow for good staining results, it also results in formation of the undesirable precipitate.
An automated H&E staining instrument consists of many parts having substantial cost. Hematein precipitate buildup on surfaces of tubing, valves, dispense manifolds, etc. can have impacts ranging from on-slide precipitate to interference or occlusion of hematoxylin dispense. Precipitation is also exacerbated by contact with metal. This is especially problematic for automated systems which contain metal parts such as nozzles and spray heads with very small diameter openings which can be clogged by precipitates. In the case of on-the-slide precipitate, the impact can be as low as being a nuisance for the pathologist reading the slide, to as high as impacting diagnostic utility. Substantial buildup of precipitate in the staining module can require the replacement of parts, or in the worst case, replacement of an entire staining module to remediate this issue.
The problem of precipitate can be solved through the use of a cleaning solution of some kind that dissolves the precipitate. The chemical makeup and physical properties of the cleaning solution must be tailored to the design of the staining module, such that the solution is compatible with the materials that comprise the various parts of the module. Additional considerations such as corrosiveness and health/safety hazards must be kept in mind from both a manufacturing and customer use point of view.
Traditional cleaning solutions for the removal of hematoxylin precipitate are generally modeled on ‘acid alcohol’, whose composition comprises a low percentage of hydrochloric acid (1-2%) in an ethanol-water mixture (typically 70% ethanol, 30% water). A solution comprised of hydrochloric acid (˜1%) in a mixture of propylene glycol and water was found to dissolve the precipitate formed from hematoxylin. The solubility of the precipitate in this solution was determined to be ˜15 milligrams/milliliter, and the dissolution rate in the absence of active mixing was quite low (empirical observation). In actual experiments with this cleaner, the contact time of the cleaner with the interior parts of the staining module may need to be on the order of days in order to effect total removal of precipitate. As a preventative maintenance cleaning solution for a staining instrument or module, the slow rate of dissolution of the precipitate by this traditional cleaner is unacceptably long. A stronger concentration of hydrochloric acid would improve both the rate of dissolution and the total solubility at the expense of being a more corrosive and hazardous material.
The use of chlorine bleach (sodium hypochlorite) to remove hematoxylin stains from affected surfaces has also been known. These bleach solutions are corrosive, however, and they can be a nuisance and a potential safety hazard to work with as they often discolor clothing and other textiles. Additionally, many people are sensitive to the chlorine fumes that emanate from these solutions.
A better solution to this issue would be in the use of a more efficacious formulation that is able to dissolve the precipitate to a higher extent and at a faster rate. Therefore, a need exists for development of a hematoxylin/hematein precipitate cleaning system and procedures that are compatible with internal storage containers, delivery lines and nozzles of automated sample processing instruments.
It is known that hydrogen peroxide is a strong oxidizer of odor causing molecules in textiles. During the treatment of affected odorous textiles hydrogen peroxide oxidizes the odorous precipitate molecules into non-malprecipitateous compounds. For example, pet urine precipitate molecules may be oxidized and rendered non-malprecipitateous. The treatment process is often combined with a cleaning device such as a vacuum cleaner. Since an aqueous solution of hydrogen peroxide is relatively stable at room temperature and hydrogen peroxide will not substantially decompose into oxygen and water unless activated, the textile odor removal process provides a buffering agent to activate the hydrogen peroxide once the two agents are mixed. In the known textile odor removal treatment the liquid buffering agent is often sodium carbonate.
Overview of the Technology
One embodiment is a method for removing hematein precipitate from internal storage containers, delivery lines, nozzles, and other reagent delivery components of automated hematoxylin staining apparatus. This can be achieved by combining a liquid oxidizing component comprising a peroxide and water and an alkaline component in a container to form a hematein precipitate removal solution having a pH in the range of between about 8.9 and 10.5. I.e., the liquid oxidizing component and the alkaline component are provided as separate components before combining them in a container. The container is then placed in a position to enable the hematoxylin dispensing components of an automated hematoxylin staining system to receive and move the solution through the interior surfaces of those dispensing components. The automated staining system is activated to move the precipitate removal solution into the interior surfaces of the hematoxylin dispensing components of the automated staining system were the interior surfaces are in contact with the precipitate removal system for at least 30 minutes.
Another embodiment relates to a precipitate removal system that includes a liquid oxidizing component comprising a peroxide and water; and a liquid alkaline component comprising a sodium citrate and water, wherein the liquid oxidizing component and the liquid alkaline component are combinable to form a solution having a pH from prior to contacting the hematein precipitate.